The relationship between aging and heart disease is well known (American Heart Association. 2000 Heart and Stroke Statistical Update. Dallas, Tex.: American Heart Association; 1999). The prevalence of heart failure is almost 70 times higher in persons 65 years of age or older, compared to persons aged 20–34 years (American Heart Association. 2000 Heart and Stroke Statistical Update. Dallas, Tex. American Heart Association; 1999). Furthermore, cardiac functional reserve declines with age in humans (Fleg J L, et al. Impact of age on the cardiovascular response to dynamic upright exercise in healthy men and women. Journal of Applied Physiology. 1995;78:890–900; Lakatta E G. Circulatory function in younger and older humans in health. In: Hazzard W R, Blass J P, Ettinger W H, Halter J B, Ouslander J G, eds. Principles of geriatric medicine and gerontology. New York: McGraw-Hill; 1999:645–660). Nearly 80% of hospital admissions in the United States for heart failure involve patients over 65 years of age (Rich M W. Heart failure. In: Hazzard W R, Blass J P, Ettinger W H, Halter J B, Ouslander J G, eds. Principles of geriatric medicine and gerontology. New York: McGraw-Hill; 1999:679–700). Due to the complexity of the human organism and its long life-span, there is a need for simple model systems for identifying genes and agents involved in cardiac function, including changes in cardiac function that are related to aging. A need exists for appropriate models for studying the aging of tissues that have very limited replicative capacity, such as the heart, which play an important role in determining human mortality.
The genome of Drosophila melanogaster was the first to be fully sequenced for an animal possessing a circulatory system (Adams M D, et al., The genome sequence of Drosophila melanogaster. Science. 2000;287:2185–2195). The heart of the fly consists of a tubular structure that contracts spontaneously throughout the insect lifespan and has the main function of circulating the hemolymph, which transports energy substrates from the abdomen to the thorax and head (Rizki T M. The Circulatory System and Associated Cells and Tissues. In: Ashburner M, Wright T R F, Eds. The Genetics and Biology of Drosophila. London: Academic Press; 1978:397–452). Investigating age-associated changes in Drosophila with a focus on cardiac function has clear advantages. By directly assessing the status of the heart, the complexity of the object of study is reduced, which should yield a smaller, more manageable, set of candidate genes for subsequent analysis after initial genetic screens. Furthermore, several of the most promising models used in aging research (yeast (Sinclair D A, Mills K, Guarente L. Molecular mechanisms of yeast aging. Trends in Biochemical Sciences. 1998;23:131–134) and recently even bacteria (Pennisi E. Evolutionary trends from bacteria to birds. Science. 2000;289:1131–1133)) are only informative for the replicative senescence of actively dividing cells.
Many important findings for human medicine and biology have originated from studies in Drosophila. Examples are the identification of genes regulating embryonic development (Nusslein-Volhard C. and Wieschaus E. 1980. Mutations affecting segment number and polarity in Drosophila. Nature 287:795–801), the initial identification of several components of the apoptotic machinery (White K. et al. 1994. Genetic control of programmed cell death in Drosophila. Science 264:677–683), and elucidation of gene pathways involved in neurogenesis (Artavanis-Tsakonas S. et al. 1983. Molecular cloning of Notch, a locus affecting neurogenesis in Drosophila melanogaster. Proc Natl Acad Sci USA 80:1977–1981).
Several groups have exploited Drosophila genetics for identifying genes regulating cardiac development in the fly, and this approach has been useful for guiding research on cardiac development in vertebrates. For example, Bodmer and Venkatesh (Heart development in Drosophila and vertebrates: conservation of molecular mechanisms. Developmental Genetics. 1998;22:181–186) reported the identification of the Drosophila gene tinman, which prompted the cloning of homologues regulating cardiac development in mice (Nkx2-5/Csx) by Lints et al. (Lints T J, et al. Nkx-2.5: a novel murine homeobox gene expressed in early heart progenitor cells and their myogenic descendants. Development. 1993; 119:419–4) and Komuro et al. (Komuro I, Izumo S. Csx: a murine homeobox-containing gene specifically expressed in the developing heart. Proceedings of the National Academy of Sciences of the United States of America. 1993;90:8145–8149). The finding of homologous genes that similarly influence development of the heart-like organ of Drosophila and the mouse heart indicates that some aspects of fly cardiac biology are common to mammals. The relevance of some fly genes to human cardiac pathology is supported by the finding, by Curran et al. (Curran M E, et al. A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome. Cell. 1995;80:795–803), that mutations in the HERG potassium channel gene cause long-QT syndrome, a potentially fatal cardiac arrhythmia (Curran M E, et al., A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome. Cell. 1995;80:795–803). HERG was first identified by virtue of its homology to the Drosophila potassium channel gene “ether-a-go-go” (Warmke J W, Ganetzky B. A family of potassium channel genes related to eag in Drosophila and mammals. Proc Natl Acad Sci USA. 1994;91:3438–3442).
Several human disease models have been developed in Drosophila, particularly for neurological diseases (Min K T, Benzer S. Preventing neurodegeneration in the Drosophila mutant bubblegum. Science. 1999;284:1985–1988; Kazemi-Esfarjani P, Benzer S. Genetic suppression of polyglutamine toxicity in Drosophila. Science. 2000;287:1837–1840; Feany M B, Bender W W. A Drosophila model of Parkinson's disease. Nature. 2000;404:394–398). Drosophila is also commonly employed as a model-organism for studying the genetics of aging, partly because it represents a genetically tractable organism with a short life span (Rose M R. Evolutionary Biology of Aging. New York: Oxford University Press; 1991). For example, a gene that controls life span in flies was identified by genetic screens (Lin Y J, Seroude L, Benzer S. Extended life-span and stress resistance in the Drosophila mutant methuselah. Science. 1998;282:943–946). The search for genes extending life span in Drosophila is actively underway and has recently begun to provide insights into the genetics of aging in this animal (Lin Y J, Seroude L, Benzer S. Extended life-span and stress resistance in the Drosophila mutant methuselah. Science. 1998;282:943–946). Mutant flies have been screened for variations in life-span, revealing that single gene mutations can increase the life-span by as much as 35% in these invertebrate animals. The problem with these methods is two-fold. First, the predominant causes of death in aged fruit flies are unknown and might be largely irrelevant to those affecting humans. Second, in rats the main causes of mortality in old animals (kidney disease and certain types of cancer) are not the same illnesses that are the most common causes of death in humans (Yu B P, et al. Life span study of S P F Fischer 344 male rats fed ad libitum or restricted diets: longevity, growth, lean body mass and disease. Journal of Gerontology. 1982;37:130–141).
Therefore fly screens based on mortality could lead to the identification of genes affecting a physiological process, maybe irrelevant to human health.
Other groups have examined fly cardiology at the cellular level. A few manuscripts from the early seventies report abnormalities in cardiac cell ultrastructure (especially involving mitochondria) in the hearts of aged flies, as studied by electron microscopy (Burch G E, Sohal R S, Fairbanks L D. Senescent changes in the heart of Drosophila repleta Wollaston. Nature. 1970;225:286–288; Burch G E, et al. Ultrastructural changes in Drosophila heart with age. Archives of Pathology & Laboratory Medicine. 1970;89:128–136). However, functional assessments of the whole heart have not been performed. Essentially nothing is known about cardiac changes that might occur with aging in the fly and attempts have not been made to exploit Drosophila genetics for investigations of adult cardiac dysfunction.
A commonly recognized limitation in the search for single gene mutations that can lengthen the lifespan of Drosophila is inbreeding depression (Tower J. Aging mechanisms in fruit flies. BioEssays. 1996;18:799–807). To make a recessive mutation homozygous and to analyze its phenotype in Drosophila requires inbreeding and this favors the fixation of alleles possessing a deleterious effect on lifespan. Working on a parameter that can be measured throughout life (heart function) should allow us to identify beneficial mutations, detecting their effect at early ages, even against an unfavorable genetic background due to inbreeding that could shorten the lifespan non-specifically.
Another advantage of using the fly as a model pertains to the size of its genome. Whereas the human genome may contain over 60,000 genes, only approximately 14,000 genes have been identified in the fly genome (Adams M D, et al. The genome sequence of Drosophila melanogaster. Science. 2000;287:2185–2195). According to Ohno's widely accepted hypothesis, two rounds of gene duplications are believed to have occurred in the human genome since the last common ancestor shared with Drosophila (Ohno S. Ancient linkage groups and frozen accidents. Nature. 1973;244:259–262). Human genes are often members of extended families with redundant functions, making genetic analysis problematic in higher eukaryotes compared to invertebrates such as C. elegans or Drosophila. Thus, approaching the problem of age-associated cardiac deterioration in a genetically tractable organism such as the fly can help to avoid genetic redundancy.
Other methods of studying the function of the fly heart are not satisfactory when compared with the present invention. For example, the method of White et al. (Effects of deuterium oxide and temperature on heart rate in Drosophila melanogaster 1992. J Comp Physiol B 162:278–83) and Johnson et al. (Modulation of Drosophila heartbeat by neurotransmitters. 1997. J Comp Physiol B 167:89–97) is limited to early pupae or larvae, not adults, and only heart rate can be measured, images are not obtained. Heart rate is not a very good measure of cardiac work if other parameters, e.g. fractional shortening, are not obtained. Using their method the animals are immersed in a drop of water and the results are therefore not physiological because respiration is impaired. The method of Nichols et al. (Regulating the activity of a cardioaccelleratory peptide. 1999. Peptides 20:1153–58) also has the limitations of measuring only heart rate and of the use of water drop immersion. With these authors' methods, images are not obtained. More importantly, with these author's methods, only relative changes from a baseline during an acute (less than 10 minutes) experiment can be reliably measured and absolute data from individuals cannot be compared, therefore these methods are not suitable for studying age-related or genetic changes.
The present inventor found that genetic screens based on age-associated differences in heart rates under the stress of elevated temperature can be exploited for identifying evolutionarily conserved genes that either accelerate or retard the rate of age-associated cardiac decline in Drosophila. Given that a recent survey has shown remarkable conservation of human genes in the fly genome, including cardiac disease-relevant genes (Fortini M E, et al. A survey of human disease gene counterparts in the Drosophila genome. Journal of Cell Biology. 2000;150:F23–F30), candidate genes identified by such genetics screens have a strong possibility of being relevant to humans.
Therefore, there is a need to determine if an age-associated decline in some aspect of cardiac performance occurs. There is a need to develop a methodology for studying the heart-like organ in intact adults. In addition, there is a need to develop methods that can quickly measure fly heart function, so that rapid screening of genes or compounds can be made. It is the object of the present invention to address these unresolved needs.